Na+/H+ exchangers of the NHE superfamily mediate the transmembrane exchange of cations with protons to regulate salt, pH and water homeostasis. We have uncovered an evolutionarily ancient subgroup of endosomal NHE that includes yeast Nhx1 and mammalian NHE6, 7 and 9. In yeast, Nhx1 localizes to the late endosome where it regulates luminal pH to control vesicle trafficking and delivery of the multivesicular body (MVB) to the vacuole for degradation. In mammals, the MVB pathway is important in HIV biogenesis, drug detoxification, erythrocyte maturation, and protein degradation. Defects in this pathway are likely to lead to lysosomal storage disorders and concomitant neurological and kidney dysfunction. Inhibitors of endosomal NHE offer a therapeutic potential to offset defects in endosome acidification seen in Dent's and Fanconi disease, and as antiviral agents. The goal of this proposal is to extend our understanding of yeast Nhx1 function and extrapolate our findings to mammalian cells. In Aim 1, we will use a combination of yeast genetics, biochemical assays of trafficking, and electron microscopy to define the precise pH-dependent step in lysosomal biogenesis. In parallel, we will test the hypothesis that NHE6 and/or NHE9 localize and function in MVB bodies in a mammalian cell culture model. In Aim 2, we will evaluate synthetic variants of exoporide, a novel amiloride analog, to find a selective inhibitor of intracellular NHE. An immediate goal of this proposal is to complete ongoing studies that seek to derive a global view of the role of cation/proton exchange by Nhx1 (Aim 3). To this end, we will continue our analysis of the genetic basis for pH regulation (pHome) and identify genes and cellular pathways that interact with Nhx1 (phenome). In Aim 4, we will assess an emerging homology model of NHE, based on the crystal structure of E. coli NhaA, using structure- bioinformatics driven mutagenesis in conjunction with phenotype screening in yeast. These studies will focus on defining the molecular basis for differences in ion selectivity and inhibitor sensitivity between the intracellular and plasma membrane subtypes of NHE, provide insight into the mechanism of transport by the NHE superfamily, and serve as a template for the design of novel NHE inhibitors. In summary, we propose a multidisciplinary approach that targets the function and mechanism of a clinically and physiologically important family of membrane transport proteins. PUBLIC HEALTH RELEVANCE This proposal targets a newly discovered but evolutionarily ancient family of ion transporters that regulate the movement of salt, water and acid equivalents across the boundaries and compartments of all cells. We plan to define the function of these proteins using parallel approaches in yeast and cultured mammalian cells, and identify new drugs using a novel screening strategy. These drugs may offer therapeutic benefits in kidney storage diseases (Dent's and Fanconi), and against envelope viruses such as HIV.